Cell culture

cell culture

The maintenance or growth of dispersed cells in a medium after removal from the body

why culture cells?

- more ethical approach

- in vitro model of cellular interactions

- in vitro model of molecular interactions

- in vitro model of cellular/microbial interactions

- use cells as a source of biological molecules for cell-free studies

examples of in vitro models of cellular interactions

- Toxicity assays

- Screening new agents for therapeutic potential

- Estimation of differential sensitivities or susceptibilities of different cell types (ex. radiation)

- Morphological & ultrastructural studies (ex. e- microscopy)

- Studies of cellular responses to various stimuli

- Studies of intercellular interactions

- Studies of complex tissues, organoids

limitations of in vitro models

! what happens in vitro does not always predict what happens in vivo

- cell culture vs. in vivo is vastly simplified system

in vitro

within a glass, observable within a test tube

in vivo

performed or taking place within a living organism

examples of in vitro models of molecular interactions

Assays of:

- DNA replication, damage, and repair

- regulation of gene expression

- protein synthesis

- enzyme activity

- receptor signaling

examples of in vitro model of cellular/microbial interactions

- Assay of cellular responses to bacterial infection

- Assay of cellular responses to fungal infection

- Assay of cellular responses to viral infection

- Assay of host immune responses to infection

- Propagation of virus

HeLa cells

human epithelial cells of a strain maintained in tissue culture since 1951 and used in research, especially in virology.

- cells taken from Henrietta Lacks

pasteurization

A process of heating food to a temperature that is high enough to kill most harmful bacteria without changing the taste of the food.

- developped by Lois Pasteur

Germ theory of disease

idea that infectious diseases are caused by microorganisms

1952 Gey discovery

establishment of a continuous cell line from human cervical carcinoma (HeLa)

cell culture incubator

used for Internal environmental control

- water jacket - temperature stability

- temperature (adjustable)

- atmosphere (CO2, N2, O2, air)

- humidity

- 37 ºC (for most mammalian cells)

horizontal laminar flow hood

- airflow keeps airborne microorganisms and particulates out of area

-> now BSCs used

BSC II cabinet

- airflow keeps airborne microorganism and particulates out of work area

AND

- prevents their escape from the work area

- offers greater operator protection

- partial containment

phase contrast microscope

light microscope that enhances contrast; useful in examining living, unstained cells

"inverted" - light source above, lens below

centrifuge

A machine that separates particles in a liquid by rapidly whirling the liquid around a central axis

what we can control: velocity + radius

- important to balance

standard benchtop centrifuge

up to 3000-4000 x g (up to 8000 RPM)

for: sedimentation of cells

highspeed centrifuge

up to ~30,000 x g (up to 17,000 RPM)

used for: sedimentation of virus

ultracentrifuge

up to 200,000 x g (or higher)

up to 60,000 RPM (or higher)

for: sedimentation of nucleic acids, sedimentation of nanoparticles

microfuge

up to 15,000 x g

for small volumes

refrigerator

4 ºC

for reagent storage

freezers

- 20 ºC or -80 ºC

storage of:

- enzymes, viable bacteria, viruses, long term reagent storage

- do colder if storing for longer

cryobiological storage system

- liquid nitrogen freezer

-196 ºC

- storable of viable eukaryotic cells

sources of tissue

- human biopsy or body fluid

- discarded human tissue

- cadaveric human organ donors

- animal tissues, embryos

preparation of primary culture from solid tissue

- disection

- enzyme digestion → cell culture

- finely chopped → primary explants

- further dissection → "organ" culture

morphology of cell division

- cell drawns in processes

- become round and highly refractile, 'glowing'

- cytokinesis begins

- cytokinesis completes

- cells grow processes

cell proliferation kinetics

- lag phase

- log (exponential) phase

- reach confluence → stationary phase

contact inhibition

a process that stops additional cell growth when cells become crowded

cells that don't divide

ex. macrophage

- terminally differentiated cells

components of typical mammalian cell culture medium

- essential amino acids: body cannot synthesize these

- vitamins: cofactors in biochemical reactions

- salts: maintain proper osmotic pressure, serve as a buffer system for pH changes

- additional components: glucose, whole serum, phenol red, antibiotics, anti-fungals, growth factors, etc.

why is phenol red in medium?

serves as a crude pH indicator

- starts as red, turns orange and yellow as pH drops

passage of adherant cell lines

- aspirate culture medium

- rinse monolayer with buffer

- add trypsin/EDTA to flask

- rap flask on hard surface

- add fresh medum

- transfer to new flask

Trypsin-EDTA

Enzyme used to detach ADHERANT cells from a culture dish

- overexposure can damage cells

propagation of non-adherent cells

- ex. activated lymphocytes, lymphoblastoid lines, etc.

- proliferate in suspension

- estimate cell density by turbidity/cell count

- passage by dilution and transfer

cryopreservation protocol

- culture medium supplemented with 10% dimethylsulfoxide (DMSO)

→ prevents ice crystal formation in cell membranes

- controlled rate freeze

DMSO

→ used when freezing cells to prevent ice crystal formation in cell membranes

major caveat with passing cells

after X passages, are the cells really the same as what you started with?

→use cells at lowest passage for experiments

patterns of proliferation of cells

- normal cells in vitro will reach a point of senescence and death around P14-P20

- transformed cell lines will continue to divide

Properties of transformed cells in culture

- immortal: proliferate indefinitely

- rapid proliferation, high mitotic index

- loss of contact inhibition

- anchorage independent growth → proliferation in soft agar

- ultimate indicator of oncogenic transformation

mitotic index

the ratio between the number of cells in mitosis to the total number of cells.

hemocytometer

Instrument used in counting blood cells

- trypan blue viability assay

common cell culture contaminants

- fungus: mold, yeast

- bacteria

- Mycoplasma

if bacteria/fungus: media will turn yellow (lower pH) and cloudy

trypan blue

a viability stain used to differentiate dead cells (blue) from living cells (clear)

automated object counter

counts cells

-> makes cell counting very easy, bust it is very costly

yeast contaminated culture

Mold Contamination

differential interference image

allows you to see bacterial contamination

Mycoplasma

- smallest bacterial genus

- lack a cell wall

- adherent to cell membranes

- do not change media color; difficult to tell if contaminated

how to avoid contamination

- sterilization of all reusable vessels/instruments

- atmosphere control via BSC , incubator

- proper PPE, gloves, lab coat, sleeve protectors

- aseptic technique

gas autoclave

Used to sterilize medical supplies and equipment that cannot be exposed to high heat, pressure and/ or steam; commonly found in specialty practices

uses ethylene oxide: highly toxic

filter sterilization

used to sterilize heat-sensitive liquids

(0.20 micron filters)

aseptic technique

- clean all items with ethanol before placing in hood

- minimize time reagent bottles/culture vessels are open

- clean work surface with ethanol before/after

- decontaminate hood with UV for ~10 min

- seal reagents with parafilm

- frequently inspect incubators/refrigerators

cell markers

technically identifiable characteristic SPECIFIC to a particular cell type of class of cell types

- cell surface or cytoplasmic protein

- particular carbohydrate moiety

- specific enzymatic activity

selection strategies

positive selection: active "capture" of cell type of interest

negative selection: elimination of cells other than those of interest

selection characteristics

- adherence characteristics

- sensitivity to injury (ex. sensitivity to hypotonic disruption)

- cell density (ex. gradient centrifugation to isolate different blood cells)

- rate of proliferation/longevity in culture

- focus formation

- selective culture media

- antibody-mediated selection methods (ex. magnetic sorting, fluorescence activated sorting)

- cloning

- antibiotic resistance

selection by cell density

- gradient centrifugation

- ex. isolation of peripheral blood mononuclear cells from blood

antibody mediated selection: magnetic

- magnetic labelling of cells

- negative selection: unwanted cells have magnet

- positive: wanted cells have magnet, then are recovered after washing

fluorescence-activated cell sorting

cloning selection strategy

used for the propagation of identical cells from a single cell

antibiotic resistant selection strategy

applies mainly to transfected cells

why use agarose gel when culturing cells?

- to screen for transformed cells

- as a mobility constraint for cells

trans-well coculture inserts

filter membrane inserts of various pore sized; inserted into a culture well

- allows you to culture two different types of cells without having them directly contact each other

ex. use to study the impact of T-cell cytokines upon nearby uninfected endothelial cells

Matrigel

a simulated extracellular matrix

perfusion culture

- the simulation of vascular shear stress

simulates the shear stress felt by vascular cells from rapid blood flow

Organoids

miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy

bioreactor

A vessel or container in which living cells or their products are used to make a product

--> mass production of products of genetically engineered bacteria or eukaryotic cells (ex. hormones, growth factors, etc.)